1. Field of the Invention
The present invention relates to the soluble low density lipoprotein (LDL) receptor, to its production and to pharmaceutical compositions containing it.
2. Description of the Background Art
Interferons (IFN) are inducible proteins that are produced by various cells and induce an antiviral state in animal cells. There are three major types of IFNs, distinguished by their antigenic properties: xcex1, xcex2 and xcex3. IFN-xcex1 and IFN-xcex2 are related proteins of 166 or 165 amino acid residues that are induced by viruses or nucleic acids and are produced by cells from various tissues, including immune cells. IFN-xcex3 is a protein of 130-143 amino acid residues which is produced by mitogen-activated T-cells and by large granular lymphocytes. The production of IFN is usually transient and it stops shortly after the inducer disappears. For a recent review of these issues, see Taylor S. L. and Grossberg S. E., 1990, Virus Research, 15, 1-26.
In addition to the three well-characterized types of interferons, there are several reports describing partially characterized species of interferons. A group of IFN-xcex1-like (IFN-xcex11) genes and pseudogenes, also known as class II IFN-xcex1 or IFN-omega was discovered and reported (Revel, M., 1984, in xe2x80x9cAntiviral Drugs and Interferon: The Molecular Basis of their Activityxe2x80x9d, Y. Becker (ed.), Martinus Neijhoff Publ., Boston, pp. 357-434; Capon, D. J. et al., 1985, Molec. Cell. Biol., 5, 768-779; Hauptmann, R. and Swetly, P., 1985, Nuc. Acid. Res., 13, 4739-4749). These are virus-induced interferons having about 172 amino acid residues which are present in the natural mixture of human IFN-xcex1 produced by leukocytes (Adolf, G. R., 1990, Virology, 175, 410-417).
Treatment of human peripheral blood mononuclear leukocytes with a mitogen resulted in production of IFN-xcex3 and a novel IFN-like substance named IFN-xcex4 (Wilkinson, M. and Morris, A., 1983, Biochem. Biophys. Res. Comm. 111, 498-503). IFN-xcex4 was found to be acid resistant and active only on human fibroblasts having chromosome-21 trisomy and not on WISH cells. It was antigenically distinct from the three known IFN types.
Acid-labile alpha-interferons were described in several publications. An acid-labile IFN-xcex1 was induced in cultures of lymphocytes from individuals who have recently received influenza vaccine, by stimulation in vitro with the influenza virus (Balkwill, F. R. et al, 1983, J. Exp. Med., 157, 1059-1063). This type of IFN was neutralized by anti-IFN-xcex1 serum and was active on Mandin Darby Bovine Kidney (MDBK) cells. The presence of such acid-labile alpha-type IFN in sera of patients with systemic lupus erythematosus was reported (Klippel, J. H. et al, 1985, Annals Rheum. Disease, 44, 104-108). An acid-labile IFN-xcex1 was produced similarly to IFN-xcex1 by Sendai virus induction of human peripheral leukocytes (Matsuoka, H. et al., 1985, J. Gen. Virol., 66, 2491-2494). Acid-labile IFN-xcex1 was spontaneously produced in cultures of peripheral blood mononuclear cells (Fischer, D. G. and Rubinstein, M., 1983, Cellular Immunology, 81, 426-434).
Another type of IFN, called IFN-epsilon, was produced by epithelial cells exposed to virus. It was produced together with IFN-xcex2 but was active on epithelial cells and not on other cell types (Jarvis, A. P. and Kosowsky, D. I., 1984, U.S. Pat. No. 4,614,651).
Among other cytokines, TNF, IL-6 and IL-1 were reported to exhibit antiviral activity (Mestan, J. et al., 1986, Nature, 323, 816-819; Wong, G. H. W. and Goedell, D., 1986, Nature, 323, 819-822; Billiau, A., 1987, Antiviral Research, 8, 55-70). TNF is produced only by immune cells and it was suggested that IL-1 and TNF exert their antiviral activity by inducing the production of IFN-xcex2 (Billiau, A., op.cit.).
Several interferon-induced proteins have been identified and some of them were shown to be instrumental in the induction of the antiviral state by IFNs. The best studied one is (2xe2x80x2-5xe2x80x2) oligo adenylate synthetase, an intracellular enzyme which polymerizes ATP into pp(A2xe2x80x2-5xe2x80x2p)nA, where n is preferably 2 or 3, but may be as long as 15 (Kerr, I. M. and Brown, R. E., 1978, Proc. Natl. Acad. Sci. USA, 75, 256-260). Such oligomers activate a latent ribonuclease (RNASE-F) which degrades ribosomal RNA and polysomes, thereby inhibiting viral and cellular protein synthesis. Another IFN-induced intracellular enzyme is a 2xe2x80x2-5xe2x80x2; phosphodiesterase which may remove the CCA terminus of tRNA, thereby leading to inhibition of protein synthesis (Schmidt, A. et al., 1979, Proc. Natl. Acad. Sci. USA, 76, 4788-4792). A third known IFN-induced intracellular enzyme is a 70 Kd protein kinase which phosphorylates the Initiation Factor eIF-2, thereby leading to inhibition of the initiation of mRNA translation into proteins (Ohtsuki, K. et al., 1980, Nature, 287, 65-67).
Other IFN-induced intracellular proteins include the nuclear IFN-Responsive Factors (IRF-1 and IRF-2) which regulate IFN-responsive genes; metallothioneis, a 56 Kd protein of unknown function in the IFN-induced antiviral state; Factor B of the alternative complement system and the murine Mx gene product, which is responsible for resistance to influenza (Reviewed in Taylor, I. L. and Grossberg, S. E., 1990, Virus Research, 15, 1-26). Other IFN-induced cell associated polypeptides were identified on 2-D gels following IFN treatment and [35S]-methionine pulsing, but these proteins were not further characterized in terms of their structure and function (Weil, J. et al., 1983, Nature, 301, 437-439). Several cell surface interferon-induced proteins were identified, including class I and II MHC antigens, IgG, Fc receptor and cytoskeletal components (Reviewed in Revel, M., 1984, in xe2x80x9cAntiviral Drugs and Interferons: The Molecular Basis of their Activityxe2x80x9d, Y. Becker (ed.), pp. 357-434, Martinus Neijhoff Publ., Boston).
Additional IFN-induced proteins that were secreted into the medium have been disclosed in the literature, such as xcex22-microglobulin, a shedded component of the cell-surface class I MHC antigens (Dolei, A. F. et al., 1981, Antiviral Res., 1, 367-373), and plasminogen activator and lymphotoxin, which were induced in lymphocytes by IFN (Jones, C. M. et al., 1982, J. Interferon Res., 2, 377-386; Wallach, D. and Hahn, T., 1983, Cellular Immunol., 76, 390-396). IFN-xcex3-treated monocytes released TNF which enhanced the overall antiviral effect (Gerrard, T. et al., 1989, J. Interferon Res., 9, 115-124). IFN-xcex3-induced proteins of molecular weight 30,000 (extracellular) and 25,000 (intracellular) were described (Luster A. D. et al., 1988, J. Biol. Chem., 263, 12036-12043), but their role was not determined.
Although many IFN-induced proteins have been disclosed, none of them is related to a soluble LDL receptor. The existence of a soluble LDL receptor as a separate protein has not been so far disclosed. The full size low density lipoprotein receptor (LDLR) is a transmembrane glycoprotein which is not soluble in the absence of detergents. It consists of 839 amino acid residues and exhibits a molecular weight of 164,000. Its only known function is to internalize LDL and VLDL. Structurally it consists of several domains, some of which are shared with other proteins. The N-terminal ligand-binding domain is made of 292 amino acid residues arranged in 7 cysteine-rich imperfect repeats. This domain is followed by a region homologous to the EGF precursor (400 amino acid residues), a region of 58 amino acid residues rich in O-linked sugars, a single trans-membrane domain of 22 amino acid residues and a cytoplasmic domain of 50 amino acid residues (Schneider W. J. et al., J. Biol. Chem. 257, 2664-2673, 1982; Yamamoto T. et al., Cell 39, 27-38, 1984). However, there is no mention of antiviral properties of the LDL receptor. The predicted nucleotide sequence (SEQ ID NO:3) of the cDNA corresponding to the LDL receptor in the mRNA, including the predicted LDL receptor amino acid sequence (SEQ ID NO:4) encoded thereby, according to Yamamoto et al (supra) is presented in FIG. 15.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.
It has been found that when human fibroblasts or epithelial cells are treated with an interferon, a protein showing antiviral activity is produced and accumulates in the supernatant of the cell cultures. This protein has now been purified to homogeneity and was identified as the soluble extracellular region of LDL receptor.
The present invention thus provides a soluble LDL new receptor protein consisting essentially of at least one replication of the soluble portion of an LDL receptor, or a mutein, fused protein, salt, functional derivative and/or active fraction thereof. The antiviral activity of the receptor protein may be conveniently determined, for example, in a system consisting of WISH amino cells and vesicular stomatitis virus (VSV) as a challenge.
The invention also provides a soluble LDL receptor corresponding to the extracellular portion (750 amino acid residues) of the LDL receptor, which is purified to homogeneity with respect to proteinaceous impurities.
The invention relates especially to the soluble LDL receptor comprising at least, but not exclusively, the ligand binding domain of the mature LDL receptor, and, more specifically, corresponding at least to amino acid residue 4 to 292-350 of SEQ ID NO:4 or any range therein, such as at about amino acid residue 313 of the mature LDL receptor, corresponding to 25 amino acid residues to 313-371, of SEQ ID NO:4 or any range therein, such as amino acid residues 25-313 of the LDLR precursor sequence. The C-terminus of a soluble LDL receptor is expected to be between amino acids 292 and 350 of the mature LDL receptor protein, or 313-371 of the LDLR precursor, such as any value therein.
The invention also relates to a soluble LDL receptor, including the amino acid sequence substantially as shown in FIG. 10.
In another aspect, the invention relates to a process for the preparation of the soluble LDL receptor, comprising treatment of suitable cells with an interferon, isolation of the soluble LDL receptor from the supernatant and purification thereof.
The invention further concerns recombinant DNA molecules comprising the nucleotide sequence coding for said protein or for its active muteins or fused proteins, expression vehicles comprising them and host cells transformed therewith and to a process for producing the soluble LDL receptor, its active muteins or fused proteins, by culturing said transformant cells in a suitable culture medium.
The soluble LDL receptor of the invention, its active muteins, fused proteins, and their salts, functional derivatives and active fractions, are for use as active ingredients of pharmaceutical compositions to protect mammals against viral infections.
The present invention also relates to methods for treating cells in mammals against viral infection by administration of an anti-viral effective amount of a pharmaceutical composition comprising a soluble LDL receptor protein of the present invention.
The present invention also relates to naturally occurring soluble LDL receptor. Such soluble LDL receptors can be purified and/or isolated from biological fluid samples, such as urine.
The invention also relates to methods for purifying soluble LDL receptor from biological fluid samples comprising isolation and/or purification of soluble LDL receptors from concentrated and/or filtered biological samples containing a soluble LDL receptor.